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Pulmonary arterial hypertension (PAH) is an intractable vascular disease characterized by a progressive increase in pulmonary vascular resistance caused by pulmonary vascular remodeling, which ultimately leads to right-sided heart failure. PAH remains incurable, despite the development of PAH-targeted therapeutics centered on pulmonary artery relaxants. It is necessary to identify the target molecules that contribute to pulmonary artery remodeling. Transient receptor potential (TRP) channels have been suggested to modulate pulmonary artery remodeling. Our study focused on the transient receptor potential ion channel subfamily M, member 7, or the TRPM7 channel, which modulates endothelial-to-mesenchymal transition and smooth muscle proliferation in the pulmonary artery. In this review, we summarize the role and expression profile of TRPM7 channels in PAH progression and discuss TRPM7 channels as possible therapeutic targets. In addition, we discuss the therapeutic effect of a Chinese herbal medicine, Ophiocordyceps sinensis (OCS), on PAH progression, which partly involves TRPM7 inhibition.

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-β2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, ‘E7K’, was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.

An organism stems from assemblies of a variety of cells and proteins. This complex system serves as a unit, and it exhibits highly sophisticated functions in response to exogenous stimuli that change over time. The complete sequencing of the entire human genome has allowed researchers to address the enigmas of life and disease at the gene- or molecular-based level. The consequence of such studies is the rapid accumulation of a multitude of data at multiple levels, ranging from molecules to the whole body, that has necessitated the development of entirely new concepts, tools, and methodologies to analyze and integrate these data. This necessity has given birth to systems biology, an advanced theoretical and practical research framework that has totally changed the directions of not only basic life science but also medicine. During the symposium of the 95th Annual Meeting of The Physiological Society of Japan 2018, five researchers reported on their respective studies on systems biology. The topics included reactions of drugs, ion-transport architecture in an epithelial system, multi-omics in renal disease, cardiac electrophysiological systems, and a software platform for computer simulation. In this review article these authors have summarized recent achievements in the field and discuss next-generation studies on health and disease. Electronic supplementary material The online version of this article (10.1007/s12576-019-00667-8) contains supplementary material, which is available to authorized users.

In the past few decades, solid evidence has been accumulated for the pivotal significance of immunoinflammatory processes in the initiation, progression, and exacerbation of many diseases and disorders. This groundbreaking view came from original works by Ross who first described that excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall is essential for the pathogenesis of atherosclerosis (Ross, Nature 1993; 362(6423): 801–9). It is now widely recognized that both innate and adaptive immune reactions are avidly involved in the inflammation-related remodeling of many tissues and organs. When this state persists, irreversible fibrogenic changes would occur often culminating in fatal insufficiencies of many vital parenchymal organs such as liver, lung, heart, kidney and intestines. Thus, inflammatory diseases are becoming the common life-threatening risk for and urgent concern about the public health in developed countries (Wynn et al., Nature Medicine 2012; 18(7): 1028–40). Considering this timeliness, we organized a special symposium entitled “Implications of immune/inflammatory responses in smooth muscle dysfunction and disease” in the 58th annual meeting of the Japan Society of Smooth Muscle Research. This symposium report will provide detailed synopses of topics presented in this symposium; (1) the role of inflammasome in atherosclerosis and abdominal aortic aneurysms by Fumitake Usui-Kawanishi and Masafumi Takahashi; (2) Mechanisms underlying the pathogenesis of hyper-contractility of bronchial smooth muscle in allergic asthma by Hiroyasu Sakai, Wataru Suto, Yuki Kai and Yoshihiko Chiba; (3) Vascular remodeling in pulmonary arterial hypertension by Keizo Hiraishi, Lin Hai Kurahara and Ryuji Inoue.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca(2+)-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca(2+)-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca(2+) elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC’s coiled-coil assembly which is essential for CaM binding. The resulting excess Ca(2+) may contribute to structural damage in the podocytes.

We describe the case a 92-year-old woman who was admitted to our hospital with choledocholithiasis and periampullary diverticulum (PAD). Due to PAD, clear visualization of the ampulla of Vater could not be obtained. Although selective bile duct cannulation was difficult, a 7-Fr plastic stent was placed during the first session. Fifteen days later, endoscopic retrograde cholangiopancreatography was retried using traction devices, and the papilla became visible. Endoscopic sphincterotomy and stone extraction were performed without any complications. The application of traction devices in endoscopic submucosal dissection may be a promising technique in cases in which endoscopic biliary intervention is difficult due to PAD.

A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5, E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 μM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 μM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the ‘WT’ or ‘E7K’ sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.

Objectives Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. Methods Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. Results VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. Conclusions We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

It is now widely accepted that advanced fibrosis underlies many chronic inflammatory disorders and is the main cause of morbidity and mortality of the modern world. The pathogenic mechanism of advanced fibrosis involves diverse and intricate interplays between numerous extracellular and intracellular signaling molecules, among which the non-trivial roles of a stress-responsive Ca2+/Na+-permeable cation channel superfamily, the transient receptor potential (TRP) protein, are receiving growing attention. Available evidence suggests that several TRP channels such as TRPC3, TRPC6, TRPV1, TRPV3, TRPV4, TRPA1, TRPM6 and TRPM7 may play central roles in the progression and/or prevention of fibroproliferative disorders in vital visceral organs such as lung, heart, liver, kidney, and bowel as well as brain, blood vessels and skin, and may contribute to both acute and chronic inflammatory processes involved therein. This short paper overviews the current knowledge accumulated in this rapidly growing field, with particular focus on cardiac and intestinal fibrosis, which are tightly associated with the pathogenesis of atrial fibrillation and inflammatory bowel diseases such as Crohn's disease.

To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto (DKT) associated with transient receptor potential ankyrin 1 (TRPA1) channels in intestinal myofibroblasts.Inflammatory and fibrotic changes were detected in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) chronic colitis model of wild-type and TRPA1-knockout (TRPA1-KO) miceChronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice. A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice. The active ingredients of DKT,The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis, and benefit inflammatory bowel disease treatment.

Background Intestinal fibrosis is a frequent complication of Crohn's disease (CD) and often leads to detrimental stricture formation. Myofibroblasts play active roles in mediating fibrotic changes in various tissues. We investigated whether transient receptor potential channels in intestinal myofibroblasts are involved in CD-associated intestinal fibrosis. Methods An intestinal myofibroblast cell line (InMyoFibs) was stimulated with transforming growth factor-β1 (TGF-β1) to model excessive fibrosis. Biopsy samples from nonstenotic or stenotic intestinal regions from patients with CD were used for quantitative comparisons of transient receptor potential channel and fibrosis-associated factor expression levels. Results TGF-β1 treatment transformed spindle-shaped InMyoFibs into filament-shaped cells with enhanced α-actin stress fiber formation, transient receptor potential canonical (TRPC) 4 and TRPC6 messenger RNA and protein expression, and basal- and agonist-induced Ca influxes. TGF-β1 also enhanced the formation of TRPC6/smooth muscle α-actin, TRPC6/N-cadherin, and TRPC4/N-cadherin coimmunoprecipitates. Inhibition of TRPC6 in InMyoFibs by RNA interference or dominant-negative mutations suppressed TGF-β1-induced Ca influxes, stress fiber formation, and smooth muscle α-actin expression, but increased COL1A1, interleukin (IL)-10, and IL-11 expression, as well as Smad-2, ERK, and p38-MAPK phosphorylation. Similar increases in phosphorylation levels were observed with TRPC and calcineurin inhibitors. In stenotic areas in patients with CD, TRPC6, ACTA2 (smooth muscle α-actin), CDH2 (N-cadherin), COL1A1, IL-10, and IL-11 were significantly increased. Conclusions These results suggest that augmented Ca influxes due to TRPC6 upregulation facilitate stress fiber formation and strengthen cell-cell interactions by negatively regulating the synthesis of antifibrotic factors in TGF-β1-treated myofibroblasts. Similar changes observed in stenotic areas of patients with CD suggest the therapeutic significance of targeting TRPC6.

Volume- and acid-sensitive outwardly rectifying anion channels (VSOR and ASOR) activated by swelling and acidification exhibit voltage-dependent inactivation and activation time courses, respectively. Recently, LRRC8A and some paralogs were shown to be essentially involved in the activity and inactivation kinetics of VSOR currents in human colonic HCT116 cells. In human cervix HeLa cells, here, inactivation of VSOR currents was found to become accelerated by RNA silencing only of LRRC8A but never decelerated by that of any LRRC8 isoform. These data suggest that LRRC8A is associated with the deceleration mechanism of VSOR inactivation, while none of LRRC8 members is related to the acceleration mechanism. Activation kinetics of ASOR currents was unaffected by knockdown of any LRRC8 family member. Double, triple and quadruple gene-silencing studies indicated that combinatory expression of LRRC8A with LRRC8D and LRRC8C is essential for VSOR activity, whereas none of LRRC8 family members is involved in ASOR activity.

BACKGROUND: Our aim was to assess whether cardiac hypertrophy is associated with cognitive function independently of office, 24-h, or sleep blood pressure (BP) levels in older hypertensive patients treated with antihypertensive medications. METHODS: In this cross-sectional study, we recruited 443 hypertensive patients aged over 60 years (mean age: 73.0 years; 41% men) who were ambulatory, lived independently, and were without clinically overt dementia. They underwent measurements of 24-h BP monitoring, echocardiographic left ventricular mass index (LVMI), and cognitive function (mini-mental state examination, MMSE). RESULTS: MMSE score was inversely associated with office, 24-h, awake, and sleep systolic BP (SBP) (each, P < 0.05). There was a close association between MMSE score and LVMI (ρ = -0.32; P < 0.001). Using multiple logistic regression analysis including numerous covariates (i.e., age, sex, obesity, current smoking, educational level, duration of antihypertensive medications, renal dysfunction, statin use, and previous history of cardiovascular disease), the odds ratio (OR) for the presence of cognitive dysfunction, defined as the lowest quartile of MMSE score (median MMSE score: 23 points; n = 115), was estimated; the presence of cardiac hypertrophy (LVMI ≥125 kg/m(2) in men and ≥110 kg/m(2) in women) as well as uncontrolled 24-h BP (mean 24-h SBP/diastolic BP (DBP) ≥130/80 mm Hg) or sleep BP (mean sleep SBP/DBP ≥120/70 mm Hg), but not uncontrolled office BP (mean office SBP/DBP ≥140/90 mm Hg), were independently associated with cognitive dysfunction (all P < 0.05). CONCLUSIONS: Among older hypertensive patients with antihypertensive medications, those who had echocardiographically determined cardiac hypertrophy may be at high risk for cognitive dysfunction, irrespective of their office BP and 24-h BP levels.

Decidualization is an ovarian steroid-induced remodeling/differentiation process of uterus essential for embryo implantation and placentation. Here, we investigated the possible involvement of enhanced Ca2+ dynamics in the decidualization process in human endometrial stromal cells (hESC) in its connection with a recently emerging nonvoltage-gated Ca2+ entry channel superfamily, the transient receptor potential (TRP) protein. Combined application of 17β-estradiol (E2) (10 nm) and progesterone (P4) (1 μm) for 7–14 d resulted in morphological changes of hESC characteristic of decidualization (i.e. cell size increase), whereas sole application of E2 exerted little effects. A 7- to 14-d E2/P4 treatment greatly increased the expression level of decidualization markers IGF binding protein-1 (IGFBP-1) and prolactin and also up-regulated the expression of TRPC1, a canonical TRP subfamily member that has been implicated in store-operated Ca2+ influx (SOC) in other cell types. In parallel with this up-regulation, SOC activity in hESC, the nuclear translocation of phosphorylated cAMP responsive element binding protein (p-CREB) and the expression of Forkhead box protein 01 were enhanced significantly. Small interfering RNA knockdown of TRPC1 counteracted the E2/P4-induced up-regulation of IGFBP-1 and prolactin and enhancement of SOC activity together with the inhibition of hESC size increase, p-CREB nuclear translocation, and FOXO1 up-regulation. Coadministration of SOC inhibitors SK&F96365 or Gd3+ with E2/P4 also suppressed the up-regulation of IGFBP-1 and hESC size increase. Similar inhibitory effects were observed with extracellularly applied TRPC1 extracellular loop 3-directed antibody, which is known to bind a near-pore domain of TRPC1 channel and block its Ca2+ transporting activity. These results strongly suggest that up-regulation of TRPC1 protein and consequent enhancement of SOC-mediated Ca2+ influx may serve as a crucial step for the decidualization process of hESC probably via p-CREB-dependent transcriptional activity associated with FOXO1 activation.

Objective— The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)–induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. Methods and Results— Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II–induced contraction was largely dependent on Ca 2+ influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)–dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II–induced Ca 2+ influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II–induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. Conclusion— PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RT‐PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (I spont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. I spont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

Rationale : Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. Objective : We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca 2+ channel known to positively regulate prohypertrophic calcineurin–nuclear factor of activated T cells (NFAT) signaling. Methods and Results : In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca 2+ influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II–induced cardiac hypertrophy in mice. Conclusions : Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

TRPタンパク質は電位変化以外の種々の物理化学刺激で活性化されるCa2+透過型非特異的陽イオンチャネルであり，感覚伝導を始め種々の生体機能とその破綻による病態に関与していると推測されている．最近の研究成果から，TRPタンパク質（チャネル）のうちの多くが，低浸透圧，ずり応力などの機械刺激で活性化されることが明らかとなってきた．興味深いことに，そのうちの多くが，血圧・血流変化等の機械刺激に暴露されている心血管系に発現している．しかし，これらの機械刺激感受性TRPチャネルがどのような分子機序によって活性化されるのか不明であった．本稿では，心血管系全般に亘って高レベルで発現し，血管緊張度，心血管のリモデリング等に密接に関与していることが明らかとなってきたTRPC6タンパク質に焦点を絞り，このチャネルタンパク質が受容体刺激，機械刺激の協働作用によって極めて効率的に活性化され，その分子機序として，PLC/ジアシルグリセロール（DAG）系とPLA2/ω-hydroxylase/20-HETE系のクロストークが重要であるという我々の仮説を中心に，心血管TRPタンパク質を巡る機械情報伝達のトピックスを紹介する．

Although Ca influx plays a pivotal role in neurohormonal and myogenic control of vascular tone and slow progressive vascular remodeling, the molecular identification of those Ca sources/pathways had long been elusive. The review will introduce recent discoveries that mammalian homologues of Drosophila transient receptor potential protein expressed in various regions of the vasculature exhibit appropriate features to elucidate these pathways, with associated dysfunctions.Recent investigations have revealed that expressed as well as native vascular transient receptor potentials behave as Ca-permeable cation channels that open in response to phospholipase C-coupled vasoconstrictors, mechanical forces and/or hypertrophic stimuli, thereby regulating the vascular resistance/tone and blood pressure/flow, and proliferative/apoptotic reorganization of vascular tissues. Notably, one vascular function relies on the coordinated interplay of multiple vascular transient receptor potential isoforms and vice versa. Imbalance of expression and altered activities of these vascular isoforms likely contribute to disorders such as hypertension, vasospasm, atherosclerosis and aneurysm.Such multifunctionality and multifaceted aspects of vascular transient receptor potentials not only suggest their unprecedented importance in regulating vascular functions, but may also offer the possibility of developing new drugs or 'calcium antagonists' that would work more selectively and elaborately for a wide range of vascular diseases based on entirely different strategies.

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (≥20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na+ and Cs+ and inhibited by Gd3+ (30 µM) and DIDS (100 µM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca2+ (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca2+-stores, first activated and then abolished SACs. Tetracaine (100 µM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca2+ and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation. Keywords:: TRPM4, stretch, cation channel, cerebral artery, smooth muscle

Despite considerable advances in the research and treatment, the precise relationship between inflammation and cardiovascular (CV) disease remains incompletely understood. Therefore, understanding the immunoinflammatory processes underlying the initiation, progression, and exacerbation of many cardiovascular diseases is of prime importance. The innate immune system has an ancient origin and is well conserved across species. Its activation occurs in response to pathogens or tissue injury. Recent studies suggest that altered ionic balance, and production of noxious gaseous mediators link to immune and inflammatory responses with altered ion channel expression and function. Among plausible candidates for this are transient receptor potential (TRP) channels that function as polymodal sensors and scaffolding proteins involved in many physiological and pathological processes. In this review, we will first focus on the relevance of TRP channel to both exogenous and endogenous factors related to innate immune response and transcription factors related to sustained inflammatory status. The emerging role of inflammasome to regulate innate immunity and its possible connection to TRP channels will also be discussed. Secondly, we will discuss about the linkage of TRP channels to inflammatory CV diseases, from a viewpoint of inflammation in a general sense which is not restricted to the innate immunity. These knowledge may serve to provide new insights into the pathogenesis of various inflammatory CV diseases and their novel therapeutic strategies.

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

It has long been known that many chemical and physical stimuli imposed on the cell from its exterior environments elicit a long-lasting Ca2+ influx through yet poorly elucidated transmembrane pathways distinct from voltage-gated and fast ligand-gated Ca2+ entry channels, thereby activating and modulating a variety of cellular functions. Recent progress in molecularly identifying these pathways, initiated from the discovery of Drosophila's visual transduction mutants transient receptor potential (TRP) proteins, has begun to reveal the presence of an enormous superfamily of non-voltage-gated Ca2+ channels. The mammalian members of TRP superfamily are (except for two members) Ca2+-permeable non-selective cation channels which are constitutively active or gated by a multitude of physicochemical stimuli such as receptor stimulation, phospholipids, oxidants, pheromones, cell volume change/shear stress, exogenous compounds affecting sensations, and changes in ambient temperature, acidity and osmolarity and cellular metabolic status. Owing to these diversities in activation and their broad distribution from brain to peripheral organs and tissues, TRP channels are now thought to be involved in divergent physiological functions including; pain and taste transductions; thermo- and mechano-sensations; regulation of mineral absorption/reabsorption; blood pressure, gut motility and airway responsiveness; cell proliferation/death, some of which seem tightly associated with specific genetic disorders. These features will render TRP channels the attractive novel molecular targets for future drug therapy. This paper briefly overviews the current knowledge available for these channels with a main interest in their possible linkage with in vivo function.

Local blood flow is critically determined by the arterial tone in which sustained Ca(2+) influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca(2+) entry channel molecules involved in this Ca(2+) influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca2+-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca2+ concentration with a parallel increase in the magnitude of spontaneous Ca2+ influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (Ispont) having Ca2+/Mg2+-inhibited but -permeating properties. Various cation channel blockers inhibiting Ispont (Gd3+, La3+, LOE908, 2-APB) suppressed the spontaneous Ca2+ influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg2+ or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of Ispont. In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced Ispont density and the magnitude of spontaneous Ca2+ influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G1/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca2+ influx pathway. Keywords:: cell proliferation, basal Ca2+ influx, transient receptor potential protein, melastatin family

Ion channels and transporters act as major components that regulate membrane excitability in neurons, muscles, and some secretory glands, but may also contribute to the regulation of proliferation, differentiation, and death in a greater variety of cells including non-excitable ones. The molecular basis of ionic mechanisms underlying the later regulation has been partly identified in the last several years and is a hot issue now. In this short review, some of the molecular mechanisms underlying these regulations and novel compounds acting on the mechanisms were introduced as exciting topics in this area. Several types of transient receptor potential (TRP), identified as Ca(2+)-permeable, non-selective cation channels, may play obligatory roles in functional complexes, which regulate multiple signal transduction pathways triggering proliferation, differentiation, or death of many cell types. In addition, the relation between Cl(-) pump activity and the induction of beta-amyloid protein toxicity for neuronal cell death in Alzheimer disease was described. Unique functions of H(+) channel and pump in osteoclasts in bone mineral homeostasis and remodeling were also discussed. Finally, topics about activation of specific types of Cl(-) channels and K(+) channels, which are responsible for the induction of apoptosis or proliferation in several types of cells, were introduced.

In both excitable and non-excitable cells, many chemical and physical stimuli elicit continuous Ca2+ influx through yet poorly understood pathways distinct from voltage-gated Ca2+ channels, leading to activation and modulation of various cellular functions. The molecular entities of these pathways have long been enigmatic, but important clues have been obtained from recent investigations on the Drosophila transient receptor potential (TRP) protein and its mammalian homologues. TRP proteins function as non-voltage-gated Ca2+ channels that are constitutively active or gated by a multitude of stimuli including light, pheromones, lipids, temperature, acid, osmolarity, and oxidative stress; and thus they may play divergent roles in cell pathophysiology. This short paper briefly overviews the current knowledge about these channels with a main focus on their possible linkage with in vivo function.

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C–insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.

Cellular stimulation from the surrounding extracellular environment via receptors and other pathways evoke activation of Ca2+-permeable cation channels that form essential signaling pathways in controlling biological responses. An important clue to understand the molecular mechanisms underlying these cation channels (tentatively termed as receptor-mediated cation channels (RMCC)) was first provided through molecular studies of the transient receptor potential (trp) protein (TRP), which controls light-induced depolarization in Drosophila photoreceptor cells. Use of the genetic information and recombinant expression technique lead to the discovery of numerous mammalian TRP homologues revealing novel RMCCs. In this review, we focus on the dramatic progress in the molecular investigation of RMCC in mammalian systems. The recent findings should provide powerful tools for the development of novel pharmaceutical targets.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

The molecular mechanism underlying Ca(2+)/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 μm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 μm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln(855)-Glu(877)) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr(487) was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr(487) (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr(487) is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg(8)-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr(487) may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.

Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7 > C6 > C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is most likely the critical event for VMI in all three channels. When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents in A7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P2 depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P2 levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P2 depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P2, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner.

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E2(PGE2) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca2+. In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca2+-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca2+influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd3+-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca2+influx by TNF-α, whereas potentiated TNF-α induced PGE2production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca2+influx, and COX-2-dependent PGE2production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE2production without affecting TRPC1 expression and the Ca2+influx. Finally, the suppression of store-dependent Ca2+influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE2production, respectively, at the downstream of TRPC1-associated Ca2+influx.

A 37-year-old man was referred to our hospital with a cough. His blood test result and chest radiography findings were normal. High resolution chest computed tomography (HRCT) showed diffuse ground glass attenuation (GGA) with centrilobular distribution in the bilateral upper lung fields. As diagnosis could not be made using transbronchial lung biopsy and bronchoalveolar lavage, video-assisted lung biopsy was performed. The histological findings were a thickening of the alveolar walls with infiltration of lymphocyte-dominant inflammatory cells, and exudation of pigmented macrophages in the air spaces of the respiratory bronchioles and alveolis. We diagnosed his illness as respiratory bronchiolitis-associated interstitial lung disease. Six months after stopping smoking, his symptoms, and the GGA on chest HRCT improved.

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

TRPC6 is a non–voltage-gated Ca 2+ entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interactions of receptor and mechanical stimulations in activating this channel using the patch clamp technique. In HEK293 cells expressing TRPC6, application of mechanical stimuli (hypotonicity, shear, 2,4,6-trinitrophenol) caused, albeit not effective by themselves, a prominent potentiation of cationic currents ( I TRPC6 ) induced by a muscarinic receptor agonist carbachol. This effect was insensitive to a tarantula toxin GsMTx-4 (5 μmol/L). A similar extent of mechanical potentiation was observed after activation of I TRPC6 by GTPγS or a diacylglycerol analog 1-oleoyl-2-acetyl- sn -glycerol (OAG). Single TRPC6 channel activity evoked by carbachol was also enhanced by a negative pressure added in the patch pipette. Mechanical potentiation of carbachol- or OAG-induced I TRPC6 was abolished by small interfering RNA knockdown of cytosolic phospholipase A 2 or pharmacological inhibition of ω-hydroxylation of arachidonic acid into 20-HETE (20-hydroxyeicosatetraenoic acid). Conversely, direct application of 20-HETE enhanced both OAG-induced macroscopic and single channel TRPC6 currents. Essentially the same results were obtained for TRPC6-like cation channel in A7r5 myocytes, where its activation by noradrenaline or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation of receptor and mechanical stimulations may synergistically amplify transmembrane Ca 2+ mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A 2 /ω-hydroxylase/20-HETE pathways.

Transient receptor potential (TRP) proteins constitute a large non-voltage-gated cation channel superfamily, activated polymodally by various physicochemical stimuli, and are implicated in a variety of cellular functions. Known activators for TRP include not only chemical stimuli such as receptor stimulation, increased acidity and pungent/cooling agents, but temperature change and various forms of mechanical stimuli such as osmotic stress, membrane stretch, and shear force. Recent investigations have revealed that at least ten mammalian TRPs exhibit mechanosensitivity (TRPC1, 5, 6; TRPV1, 2, 4; TRPM3, 7; TRPA1; TRPP2), but the mechanisms underlying it appear considerably divergent and complex. The proposed mechanisms are associated with lipid bilayer mechanics, specialized force-transducing structures, biochemical reactions, membrane trafficking and transcriptional regulation. Many of mechanosensitive (MS)-TRP channel likely undergo multiple regulations via these mechanisms. In the cardiovascular system in which hemodynamic forces constantly operate, the impact of mechanical stress may be particularly significant. Extensive morphological and functional studies have indicated that several MS-TRP channels are expressed in cardiac muscle, vascular smooth muscle, endothelium and vasosensory neurons, each differentially contributing to cardiovascular (CV) functions. To further complexity, the recent evidence suggests that mechanical stress may synergize with neurohormonal mechanisms thereby amplifying otherwise marginal responses. Furthermore, the currently available data suggest that MS-TRP channels may be involved in CV pathophysiology such as cardiac arrhythmia, cardiac hypertrophy/myopathy, hypertension and aneurysms. This review will overview currently known mechanisms for mechanical activation/modulation of TRPs and possible connections of MS-TRP channels to CV disorders.

We investigated the inhibitory role of the nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by approximately 70%) by a NO donor SNAP (100 microm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 microm) or after G-protein activation with intracellular perfusion of GTPgammaS (100 microm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 microm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of (32)P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg(8)-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO-cGMP-PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

Aim: The aim of the present study was to explore the mechanism for the Ca 2+ -dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. Method: The whole-cell patch-clamp technique was used in the study. Results: With Ca 2+ -free external solution, the perfusion of 100 μmol/L carbachol to, or dialysis of the cell with 100 μmol/L guanosine 5'-3- O -(thio)triphosphate (GTPgS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca 2+ into the bath, and recovery from this inhibition was only partial after the Ca 2+ removal, unless vigorous intracellular Ca 2+ buffering with 10 mmol/L 1,2 bis (2-aminophenoxy)ethane- N,N,N’,N’ -tetraacetic acid (BAPTA) (plus 4 mmol/L Ca 2+ ) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 μmol/L) did not undergo the inhibition persisting after Ca 2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP 3 ; 100 μmol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca 2+ -mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP 3 . Conclusion: The present results suggest that Ca 2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP 3 are essentially involved.

A 39-year-old man was referred to our hospital with anterior chest discomfort, dry cough and shortness of breath. His blood test revealed mild inflammatory change and high serum KL-6 levels. Chest radiograph and computed tomography (CT) showed ground glass attenuation with volume loss in both lower lung fields, and in particular a reversed halo sign was shown on high-resolution CT (HRCT). As transbronchial lung biopsy and bronchoalveolar lavage did not enable a diagnosis, video-assisted thoracic surgery was performed. The histological findings of the resected specimen showed cellular nonspecific interstitial pneumonia. This suggested the possibility of collagen vascular disorder (CVD) associated with interstitial pneumonitis, but no criteria of CVD were fulfilled. Although the reversed halo sign is relatively specific for cryptogenic organizing pneumonia, we report a case of cellular nonspecific interstitial pneumonia showing this sign on chest HRCT.